The effect of the troponin-tropomyosin complex on the binding of S-1 to actin has provided evidence for two different conformations of the acto.S-1, depending on the nucleotide bound to S-1. In the presence of ADP or the nucleotide analog, AMP-PNP, S-1 binds to regulated actin tightly and with positive cooperativity. However, under the same conditions, the binding of S-1 to regulated actin in the presence of ATP is much weaker and appears non-cooperative. By examining the binding of S-1.ATP and S-1.AMP-PNP to regulated actin under conditions where they bind with equal affinity to actin, we found that S-1.AMP-PNP binds cooperatively to regulated actin, whereas S-1.ATP does not. These results suggest that the structure of the acto.S-1 complex is different with ATP and AMP-PNP. We have further shown that a stable analog of the S-1.ATP state can be formed by cross-linking two cysteine groups of the S-1 molecule with N,N'-p-phenylenedimaleimide. As does S-1.ATP, the cross-linked S-1 binds very weakly to actin and does not exhibit significant cooperativity in binding to regulated actin.